Charge determination of proteins with polyelectrolyte titration

Area of Science:

• Biochemistry
• Biophysics
• Analytical Chemistry

Background:

• Determining the number of charged residues on proteins is crucial for understanding their behavior.
• Existing methods may have limitations in accuracy or accessibility.
• Polyelectrolyte complex formation is a known interaction with potential for quantitative analysis.

Purpose of the Study:

• To apply a novel photometric polyelectrolyte titration method for quantifying charged residues on globular proteins.
• To investigate the stoichiometry of protein-polyelectrolyte interactions across a physiological pH range.
• To assess the role of intramolecular salt linkages in protein structure and surface charge.

Main Methods:

• Utilized a photometric polyelectrolyte titration technique.
• Incubated protein solutions with excess oppositely charged polyelectrolytes (potassium polyvinylsulfate, polydiallylammonium chloride, N-methylglycolchitosan iodide).
• Performed back-titration with o-toluidine blue for endpoint detection to quantify residual polyelectrolyte.

Main Results:

• The titration method accurately determined the number of charged residues on various proteins (ribonuclease A, trypsin, chymotrypsin A, pepsin, cytochrome c) between pH 2 and 9.
• Protein-polyelectrolyte interactions were found to be stoichiometric, occurring via 1:1 ion pair formation.
• Experimental net charge data closely matched calculated values, suggesting significant neutralization by intramolecular salt bridges in native protein structures.

Conclusions:

• Photometric polyelectrolyte titration provides a straightforward method for determining protein surface charge.
• The findings support the importance of electrostatic cooperative interactions in biological systems.
• Intramolecular salt linkages play a significant role in neutralizing charges within native protein structures.